The present invention relates generally to a light microscope and to a method of microscopy.
A light microscope of the generic type has a sample plane, in which a sample to be examined is positionable, a light source for emitting illumination light, optical imaging means for guiding the illumination light into the sample plane, and a detector device for detecting sample light coming from the sample, wherein adjacent detector elements are at a distance from one another which is smaller than an Airy disk produced by a point in the sample plane on the detector device. Electronic means can determine an image of the sample on the basis of the detected sample light.
In a microscopy method of the generic type, for examining a sample positioned in a sample plane of a light microscope, illumination light is guided into the sample plane, the illumination light is moved as illumination scanning movement over the sample plane, and sample light coming from the sample is detected by means of a detector device having a plurality of detector elements. Adjacent detector elements are at a distance from one another which is smaller than an Airy disk produced by a point in the sample plane on the detector device. In this case, electronic means can determine an image of the sample on the basis of the detected sample light.
In the case of such light microscopes and microscopy methods a fundamental aim is that of generating a sample image with the highest possible resolution and the best possible signal-to-noise ratio.
For this purpose, the light microscope of the generic type and the microscopy method make use of detector elements which are smaller than an Airy disk produced by a point in the sample plane on the detector device.
The Airy is defined by means of the first zeros of the rotationally symmetrical light distribution of a diffraction-limited illumination spot. An Airy is thus an extent of a diffraction disk in an image plane which is brought about by a point in the sample plane. The extent can be defined as the distance between the first zeros of the diffraction disk. A diffraction-limited light distribution having the size of an Airy has a radius of 0.61 λ/NA. In this case, λ, is the light wavelength and NA is the numerical aperture.
Expediently, the distance between adjacent detector elements can be less than half or one third of an Airy disk. As a result, a point of the sample plane is always imaged onto a plurality of adjacent detector elements.
Insights regarding what measures can achieve an increase in resolution here go back to C. Sheppard and are described in the article “Super-resolution in Confocal Imaging” by Colin Sheppard et al., published in Optik 80, No. 2, 45 (1988). For increasing the resolution in the sample image, in this case image recording is followed by resorting and computation of the image data by means of a special algorithm, which is also referred to as accumulation of displaced sub-Airy detector values.
Such a method is explained with reference to FIG. 1, which schematically illustrates a sample along the x-axis of a sample plane. The sample has a sample point 42 or a fluorescent object 42. An illumination spot 44 is also illustrated. The intensity I thereof is specified on the ordinate. The dimensions of the illumination spot 44 can be diffraction-limited and are larger than the object 42 in the x-direction. If the illumination spot 44 impinges on the object 42, the latter is excited to fluorescence and emits sample light which can be detected by a detector device.
FIG. 1 furthermore illustrates an imaging, here infinitely sharp, of such a detector device 60 into the sample plane. The detector device 60 comprises a plurality of detector elements 63, 64. The latter not only receive sample light which emerges from a point of the sample plane, but an extensive receiving region is imaged onto each detector element, said region being determined by the PSF (point spread function) of the imaging. The PSF for the detector element 64 is illustrated as a dashed curve 46. The dimensions of the illumination spot 44 can likewise be determined by a PSF of a point light source.
The measured light intensity of a specific detector element 64 is then determined by a total PSF, which is the product of the PSF with regard to the illumination spot 44 and the PSF 46 with regard to the detector element 64. The maximum of the total PSF lies approximately centrally between the illumination spot 44 and the PSF 46 of the respective detector element 64. In the example illustrated, the detector element 64 therefore receives light principally from a location 61A lying centrally between the illumination spot 44 and the PSF 46. By contrast, the detector element 64 measures hardly any light from the position 61D, even though the associated PSF 46 has its maximum at said position.
For the purpose of scanning the sample, the illumination spot is then displaced from the position 44D to 44B, for example. This is designated as illumination scanning movement in the present case. The total PSF of the detector element 64 shifts as a result. Said detector element then no longer measures light from principally the position 61A, but rather 61B.
This circumstance can be used for increasing the resolution. For this purpose, the detector elements with regard to each position of the illumination spot 44 are read. The sample light signals measured in this case are assigned to different sample regions depending on the position of the illumination spot 44. That is to say that the sample light signals measured by one and the same detector element are resorted depending on the position of the illumination spot 44.
The resorting is illustrated by the curved arrows. Accordingly, a signal of the detector element 64 is assigned to the location 61A of the object 42 if an illumination spot is situated at the location 44D. Analogously, a signal of the detector element at the location 61C is assigned to the location of the object 42 in the case of an illumination spot at the location 44C. Moreover, a signal of the detector element 61B is assigned to the location of the object 42 in the case of an illumination spot at the location 44B.
An improvement of the resolution can be achieved in this way. The apparatus outlay for achieving this resorting is high, however. In addition, a time requirement for calculating the resorting is comparatively high.
The improvement of the resolution can also be described as greater weighting of the higher spatial frequencies in the optical transfer spectrum of a single-spot system. Since the light distribution within a 1-Airy pinhole diameter is used for the image generation, more photons can be detected. The signal-to-noise ratio is thus improved.
Comparable microscopes that use detection with sub-Airy resolution are described in EP 2 520 965 A1 and in York et al., Nature Methods Vol. 9, 749-754 (2012). A multi-spot illumination is additionally used. In this case, each light spot is scanned successively over different sample regions. In this regard, although an increase in speed can be achieved during the scan it is necessary to read out images recorded in each case by the detector device for different scan positions and to compute them as described previously, see for example “supplementary note 1” of the article by York et al. As a result, the image recording speed is reduced, which is disadvantageous particularly for the imaging of living cells. Moreover, computation and/or motion artefacts can arise in the image.
In order to examine a sample with increased resolution, structured illumination microscopy (SIM) has additionally become established. This uses structured illumination light which can be generated by line gratings, for example.
In the case of a laser scanning microscope (LSM), an illumination spot is used as structured illumination. Here an increased resolution is achieved by means of a confocal imaging for which a pinhole, that is to say a pinhole stop, is positioned in or on an image plane. In the case of an LSM, however, the signal-to-noise ratio is comparatively low since only a comparatively small proportion of light is used.
For simultaneously examining a plurality of sample regions, it is possible to use a microscope with a Nipkow disk. The latter comprises a plurality of pinholes arranged as Archimedean spirals. Such microscopes are described in U.S. Pat. No. 5,428,475 A and US 2008/0218849 A1.
As a result of the Nipkow disk being arranged in the common illumination and detection beam path, off-focus light is filtered. A rapid image recording can be achieved with this comparatively simple construction by rotation of the Nipkow disk. The latter is therefore also referred to as a spinning disk. The simultaneous transillumination of a plurality of pinholes of the Nipkow disk, a so-called multi-spot examination, can accelerate the sample examination further. A microscope with this construction is described in EP 1 359 452 A1. In order to guide a greater proportion of the illumination light through the pinholes of the Nipkow disk, a micro-focusing lens disk is used in this case. The latter is rotated jointly with the Nipkow disk. Sample light is likewise guided through said micro-focusing lens disk and is subsequently guided by means of a further micro-focusing lens disk in the direction of a detector.
The range of the optical limit resolution can be achieved only with a poor signal-to-noise ratio in the case of such known spinning disk microscopes.